Objective To establish a non-labeled real-time PCR assay targeting OmpA-VS2 for detection of Chlamydia trachomatis (C. trachomatis). Methods Two pairs of universal primers were designed to amplify the OmpA-VS2 region of fifteen genotypes of C. trachomatis by the nested real-time PCR. The sensitivity and specificity of the assay were analyzed and further validated with clinical positive specimens. Results All 15 genotypes of C. trachomatis were successfully amplified with a product of 168bp. The detection limit with the assay was 1 copy per reaction mixture and no cross reactions with 10 other urogenital pathogens or commensals were found. The clinical validation reveals a coincidence with the results detected by plasmid PCR. Conclusion The assay is characterized with high sensitivity and specificity, and no separated post-PCR analysis is needed. It would be an appropriate assay to be used for detection of chlamydial infection in clinical practice as well as in disease control programs.